The invention relates to methods and compositions for the elimination of T cells that recognize specific preselected targets. In particular, the invention provides methods and compositions for the treatment of graft rejection and autoimmune diseases. In one embodiment, the invention provides modified killer cells that bear a preselected target molecule (e.g. an MHC molecule) attached to a signal transduction molecule (e.g. the zeta (xcex6) chain of the T cell receptor or the low-affinity receptor for IgG, Fcxcex3RIII (CD16)). Recognition of the preselected molecule by a T cell activates the killer cell through the signal transduction molecule and the activated killer cell then kills or inhibits the T cell.
Autoimmune diseases are pathological states mediated by an undesired immune response. More than 30 autoimmune diseases are presently known; these include many which have received much public attention, including myasthenia gravis (MG) and multiple sclerosis (MS). Characteristic of these diseases is the attack by the immune system on the tissues of the victimxe2x80x94these tissue antigens being non-immunogenic in non-diseased individuals because of the tolerance of the immune system to xe2x80x9cself.xe2x80x9d In autoimmune diseases, this tolerance apparently is compromised, and the tissue of the afflicted subject is treated as an invaderxe2x80x94i.e., the immune system sets about destroying this presumed foreign target.
Similarly in graft rejection the recipient of a graft or organ transplant mounts an immune response against the foreign (non-self) tissue. In this case the response is not a loss of tolerance, but rather a legitimate, although undesired, response directed against a foreign tissue.
A crude approach to treating graft or organ rejection or autoimmune disease is, of course, general immunosuppression. This has the obvious disadvantage of crippling the ability of the subject to respond to infectious organisms or tumors. An only slightly more sophisticated approach relies on the removal of antibodies or immune complexes involving the target tissue. This also has adverse side effects, is difficult to accomplish, and is not particularly effective.
Various approaches have been attempted to interdict the immune response to specific antigens. For example, the autoantigen thyroglobulin has been conjugated to ricin A and the conjugate was shown to suppress specifically the in vitro antibody response of lymphocytes which normally respond to this antigen. It was suggested that such immunotoxins would specifically delete autoantibody-secreting lymphocyte clones (Rennie, et al., Lancet (Dec. 10, 1983) 1338-1339).
Diener, et al., Science 231: 148-150 (1986) suggested the construction of compounds which cause antigen-specific suppression of lymphocyte function by conjugating daunomycin to the hapten (in this case, of ovalbumin) using an acid-sensitive spacer. The conjugate caused hapten-specific inhibition of antibody secretion by B lymphocytes in vitro and in vivo. A conjugate of daunomycin (with an acid-sensitive spacer) to a monoclonal antibody-specific to T cells also eliminated the response by T-lymphocytes to concanavalin A.
Steerz et al., J. Immunol. 134: 841-846 (1985) utilized radiation as the toxic element in a toxin conjugate. Rats were administered a radioactively labeled, purified receptor from electric fish, prior to injection with cold receptor. Injection with this receptor is a standard procedure to induce experimental autoimmune myasthenia gravis (EAMG). Control rats that received preinjection only either of cold receptor or radiolabeled albumin, prior to administration of receptor to induce the disease develop the symptoms of EAMG; those pretreated with radioactively-labeled receptor showed reduced symptoms. It was surmised that the labeled, and therefore destructive, receptor selectively eliminated immunocompetent cells. Similar work utilizing a ricin/receptor conjugate for pretreatment was reported by Killen, et al., J. Immunol. 133: 2549-2553 (1984).
A less specific approach which results in the destruction of T cells in general is treatment with an IL-2/toxin conjugate as reported by Hixson, Medical Tribune, (Jan. 28, 1988) 4-5. In a converse, but related, approach Liu et al., Science 239: 395-397 (1988), report a method to xe2x80x9clink upxe2x80x9d cytotoxic T cells with a desired target, regardless of the cytotoxic T cell specificity. In this approach, antibody specific to the universal cytotoxic T-lymphocytes to destroy human melanoma cells when melanocyte-stimulating hormone was the hormone used.
Recent experiments have shown that, under certain circumstances, anergy or nonresponsiveness can be induced in autoreactive lymphocytes (see, Schwartz, Cell 1073-1081 (1989)). In vitro experiments suggest that antigen presentation by MHC Class II molecules in the absence of co-stimulatory signals induces a state of proliferative non-responsiveness in syngeneic T cells (Quill et al., J. Immunol. 138: 3704-3712 (1987)). These reports, however, provide no clear evidence that induction of anergy in vivo is possible or that autoimmune disease or graft rejection can be effectively treated in this manner.
This invention provides for a method of selectively inhibiting or killing T cells that recognize a specific preselected target molecule. The invention provides modified killer cells that bear a signal transduction molecule (e.g. the zeta (xcex6) chain of a T cell receptor) to which is attached the preselected target molecule (e.g. the extracellular domain of a particular MHC molecule). Recognition of the preselected molecule by a T cell activates the killer cell which then kills or inhibits the T cell. Where the preselected molecule is an extracellular domain of an MHC from a xenograft or an allograft, treatment of the graft recipient with the modified killer T cells delays or inhibits graft rejection. Similarly where the preselected molecule is an MHC that binds the antigenic determinant of the autoimmune disease, treatment of the organism with the modified killer T cells mitigates the autoimmune response directed against that antigenic determinant.
This invention provides both for methods and for compositions for the selective inhibition or destruction of T cells that specifically recognize a preselected target molecule. Thus, in one embodiment, this invention provides a method of inhibiting a T cell that specifically recognizes a preselected target molecule. The method involves contacting the T cell with a killer cell comprising a signal transduction molecule attached to a preselected target molecule that is recognized by the T cell. Suitable killer cells include, but are not limited to CTLs, NK cells, macrophages, or the precursors of these cells (e.g. hematopoietic stem cells). The signal transduction molecule may be chemically conjugated to the target molecule, or the transduction molecule/target molecule chimera may be recombinantly expressed as a fusion protein. Preferred signal transduction molecules include the xcex6 chain of a T cell receptor, the xcex3 chain of an FC receptor, the xcex7 chain, the xcex5 chain, and the xcex4 chain, rFCxcex5R1xcex2, EBV-LMP2A, BLV gp30, TAMs and ARAMs, with the xcex6 chain being most preferred.
The killer cell may be an cell that is cytotoxic to other cells when it is activated. Preferred killer cells include cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells.
Preferred target molecules include the extracellular domains of an MHC molecule, more preferably a human MHC molecule (HLA), or the extracellular domains of an MHC molecule (e.g. HLA) complexed with an antigenic determinant of an autoimmune disease (e.g. human type II collagen, or myelin basic protein NBP) or with other peptides.
In a particularly preferred method, the preselected target molecule is the extracellular domain of an MHC molecule of an allograft or a xenograft transplanted to a recipient and the killer cell is administered to the recipient where it inhibits T cells that recognize the MHC molecule and thereby reduces or eliminates a graft rejection response. Administration may be by administration of the killer cell or by administration of a precursor cell (e.g. hematopoietic stem cell) that gives rise to the killer cell. Any of the above-identified signal transduction molecules are suitable, with the xcex6 chain of the T cell receptor being preferred. In a preferred embodiment, the target molecule and the signal transduction molecule are expressed as a fusion protein.
In another particularly preferred method, the preselected target molecule is an MHC molecule that presents an antigenic determinant of an autoimmune disease and the killer cell is administered to an organism exhibiting said autoimmune disease where the killer cell inhibits T cells that recognize the antigenic determinant and reduces or eliminates an autoimmune response. Administration may be by administration of the killer cell or by administration of a precursor cell (e.g. hematopoietic stem cell) that gives rise to the killer cell. Any of the above-identified signal transduction molecules are suitable, with the xcex6 chain of the T cell receptor being preferred. In a preferred embodiment, the target molecule and the signal transduction molecule are expressed as a fusion protein. The method may additionally involve loading the MHC component of the chimera with the antigenic determinant. This may be accomplished by expression of the antigenic determinant as a fusion with the MHC/signal transduction chimera, by chemical conjugation of the antigenic determinant with the MHC, or by simple exposure of cells bearing the MHC/signal transduction chimera to the antigenic determinant before administration to an organism.
In another embodiment, this invention provides a method of prolonging the life of a cell of a tissue in a host having a T cell that specifically recognizes the tissue and mediates an immune response directed against said tissue. The method involves administering to the host an amount of a killer cell comprising an MHC molecule attached to a signal transduction molecule sufficient to inhibit the T cells that specifically recognize the tissue. The tissue may be an allograft or a xenograft. Preferred allografts or xenografts include, but are not limited to, (transplant) organs such as heart, kidney, or liver or other tissues such as skin. The MHC molecule is preferably the extracellular domain of an MHC characteristic of the allograft or a xenograft. As indicated above, suitable signal transduction molecules include the zeta xcex6 chain of a T cell receptor, the xcex3 chain of an FC receptor, the xcex7 chain, the xcex5 chain, and the xcex4 chain, rFCxcex5R1B, EBV-LMP2A, BLV gp30, TAMs and ARAMs. The chimeric MHC/signal transduction molecule may be expressed as a fusion protein. The administered cells include, but are not limited to CTLs, NK cells, macrophages, the precursors of these cells (e.g. hematopoietic stem cells), or cells transformed to express a cytotoxic activity against other cells when activated.
In still another embodiment, this invention provides a method of improving activation of a T cell by a presenting B cell. The method involves contacting the T cell with a B-cell comprising a signal transduction molecule attached to an extracellular domain of a major histocompatibility complex (MHC) molecule. Any of the above-identified signal transduction molecules are suitable, with the xcex6 chain being most preferred. A human MHC molecule (HLA) is preferred. The attachment may be by chemical coupling or fusion with fusion being most preferred. The method may be used to treat organisms exhibiting a tumor or a parasitic infection. Treatment involves administering the above-described B-cells (or precursors of these cells) to the organism in an amount sufficient to ameliorate the tumor or parasitic infection.
In still yet another embodiment this invention provides for nucleic acids encoding an extracellular domain of an MHC molecule fused to a signal transduction molecule. Preferred signal transduction molecules include xcex6, xcex3, xcex7, xcex4, and xcex5, with xcex3, xcex7 and xcex5 being more preferred and xcex6 being most preferred. Preferred MHC molecules are human MHC molecules (HLA).
This invention also provides a method of inducing immune tolerance of a preselected target molecule. The method involves providing a cell transfected with a nucleic acid encoding the target molecule operably linked to a T-cell specific promoter and administering that cell to a mammal in which tolerance is to be induced. The cell is preferably a cell that matures or differentiates in the thymus. When the cell matures or differentiates in the thymus, it expresses the target molecule where it mediates deletion or anergy of developing T cells reactive to the target molecule. The cell is preferably a bone marrow cell, more preferably a hematopoietic stem cell. The promoter is preferably a promoter that induces expression of a nucleic acid under its control (operably linked) predominantly (more preferably exclusively) when the cell containing the promoter is in the thymus. Particularly preferred promoters include a CD2 promoter, an lck promoter, a CD3 promoter, and a CD4 promoter, with an lck promoter and a CD4 promoter being most preferred.
Finally, this invention also provides for pharmacological compositions and for kits for the practice of the above described methods. Preferred pharmacological compositions include a pharmacologically acceptable excipient and a cell comprising an extracellular domain of a major histocompatibility complex (MHC) molecule attached to a signal transduction molecule. In a preferred embodiment, the MHC is fused to the signal transduction molecule. The pharmacological composition may include any of the above-identified signal transduction molecules with xcex3, xcex7 and xcex5 being more preferred and xcex6 being most preferred. Suitable cells include cytotoxic T lymphocytes, natural killer (NK) cells, B cells and precursors of any of these cells (e.g. hematopoietic stem cells).
Kits for the practice of this invention include a container containing any of the cells comprising the above-described pharmacological compositions. The kits may additionally or alternatively include expression cassettes encoding a HLA or MHC/xcex6 chimera, an HLA or MHC under the control of a T cell promoter (e.g., CD2 promoter, lck promoter, CD4 promoter, etc.), or encoding a xcex6 chain and a restriction site for the easy insertion of a nucleic acid encoding a particular target. The kit may also include grafts (e.g., tissues or organs) expressing the HLA or MHC encoded by the HLA or MHC/xcex6 construct found in the kit. In addition, the kit may include such items as means for administering the cells, means for expanding the cells ex vivo, various buffers and reagents for the culture and administration of the cells, and means (e.g., vectors) for transforming cells to express the signal transduction molecule/MHC molecule chimeras described above.
As used herein the term xe2x80x9ckiller cellxe2x80x9d refers to a cell that has cytotoxic activity and is thus capable of damaging or killing other cells with which it comes in contact. Killer cells are well known to those of skill in the art and include, but are not limited to natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), activated macrophages, and the like. The term killer cells, as used herein also includes cells that have been altered such that, when activated, they manifest a xe2x80x9ckillerxe2x80x9d phenotype, again characterized by the ability to damage or kill other cells.
A xe2x80x9cchimeric moleculexe2x80x9d is a molecule comprising two or more molecules that exist separately in their native state are but are joined together to form a single molecule having the desired functionality of all of its constituent molecules.
The terms xe2x80x9cpreselected target moleculexe2x80x9d or xe2x80x9cpreselected target polypeptidexe2x80x9d refer to molecules that are recognized by the T cell it is desired to inhibit or destroy. xe2x80x9cPreselectionxe2x80x9d refers to the fact that the identity of the target molecule is selected or known prior to the construction and/or use of cells bearing the target molecule-signal transduction molecule chimera. Preferred preselected target molecules are polypeptides.
The terms xe2x80x9cMHC polypeptidexe2x80x9d, xe2x80x9cMHC moleculexe2x80x9d or xe2x80x9cmolecule of the MHC complexxe2x80x9d, as used herein, refer to a single chain or double chain MHC protein (e.g., the xcex1 or xcex2 chain of Class II molecules or the heavy chain of Class I molecules) which may constitute all or part of the extracellular portion of the MHC complex (i.e., that portion of the MHC that is accessible to a T cell) which is in other than its native state, for example, not associated with the cell membrane of a cell that normally expresses that MHC.
The xe2x80x9cextracellular domainxe2x80x9d of class I MHC molecules involves the alpha one, two and three domains, while the extracellular domain of class II MHC molecules involves the alpha one and two domains of the alpha chain and the beta one and two domains of the beta chain. As used herein the xe2x80x9cextracellular domainxe2x80x9d of an MHC refers both to the native extracellular domains describe above as well as to modified or chemically synthesized extracellular domains. Modified extracellular domains include, but are not limited to MHC domains in which conservative substitutions are made for various amino acids, side chains of various amino acids are modified (e.g. to improve peptide presentation), or amino acids are added or eliminated (e.g. to provide the minimal molecule necessary for presentation).
The term xe2x80x9cspecifically recognizesxe2x80x9d or xe2x80x9cT cells specific for a preselected target moleculexe2x80x9d refers to a binding reaction which is determinative of the presence of a particular antigen specific T cell in the presence of a heterogeneous population of T cells and other biologics. Thus, under designated immunoassay conditions, the specified T cells bind to particular pre-selected target molecule (e.g. an allogenic MHC) or to a MHC:target molecule complex and do not bind in a significant amount to other target molecules or to other MHC:peptide complexes present in the sample. T cells specific for a preselected target molecule refers to those T cells in a heterogenous population of T cells which bind to and are selectively activated by a particular pre-determined target molecule or to a MHC:target molecule complex.
The phrase xe2x80x9cMHC capable of presenting a polypeptide (antigenic determinant)xe2x80x9d refers to an MHC molecule that binds to a polypeptide (e.g. where the polypeptide is an antigenic determinant of an autoimmune disease) and presents that polypeptide on a cell surface such that it may be specifically recognized by a T cell or a CTL.
The term xe2x80x9callograftxe2x80x9d refers to grafts between genetically different members of the same species. In humans, most organ grafts from one individual to another are allografts unless an identical twin is available as a donor. Because an allograft is genetically dissimilar to the host it is often recognized as foreign by the immune system and is rejected in an allograft reaction. xe2x80x9cXenograftsxe2x80x9d are grafts between different species such as the graft of a baboon heart to a human.
The phrase xe2x80x9cinhibiting a T cellxe2x80x9d refers to prevention of activation and proliferation of a T cell or to the actual destruction (killing) of a T cell. It is recognized that a killer cell (e.g. CTL) of the present invention, when recognized by the T cell may not always kill the recognizing T cell. However, even damage to the recognizing T cell may lead to an inability of that T cell to proliferate or otherwise participate in an immune response. Thus, inhibition, as used herein when referring to inhibiting a T cell also includes a reduction or elimination of the ability of a T cell to participate in an immune response. Alternatively, the inhibition can be mediated by induction of a cytokine profile that downregulates the activation and/or function of the T cells. Thus, for example, T cells recognizing Dd xcex3 on Th cells may induce secretion of IL-10 or owher down-regulator cytokines which do not eliminate the recognizign cell, but rather downregulate its function.
An xe2x80x9cundifferentiated cellxe2x80x9d refers to a cell that has not yet acquired the characteristics of the mature cell type. Thus, for example, hematopoietic stem cells are undifferentiated cells.